PROJECT SUMMARY Reactive oxygen species derived from NADPH oxidases and inflammation have been implicated in most vascular diseases, but also play a role in normal physiology and vascular healing via regulation of smooth muscle cell (SMC) proliferation and matrix secretion. Our laboratory has recently discovered a novel p22phox binding protein, polymerase delta interacting protein-2 (Poldip2), which regulates Nox4, polymerase-? and cell cycle proteins and has important roles in growth, migration and collagen secretion. In this proposal, we test the hypothesis that the multiple roles of Poldip2 in vascular cells are a function of its binding partners, and that these functions can be specifically targeted using small interfering peptides. In Aim 1, we plan to characterize the physical interaction between Poldip2 and p22phox with the goal of defining peptides that can be used to specifically disrupt this interaction therapeutically. Truncation mutants and pulldown assays will be used to define interacting regions and to design peptides for cell and animal delivery. Aim 2 is devoted to using these peptides to determine the mechanism by which Poldip2 regulates extracellular collagen levels. We will test the hypothesis that depletion of Poldip2 reduces autophagy, thus enhancing collagen secretion. In Aim 3, we will determine the specific consequence of smooth muscle Poldip2-mediated matrix regulation on arterial stiffness and aneurysm formation using Poldipflox/flox animals crossed with smooth muscle-specific Cre mice in both chronic and acute settings, as well as hydrogel- or microparticle-mediated delivery of peptide decoys. Together, the experiments described in this proposal will not only extend our understanding of the vascular biology of Poldip2, but will allow us to take the first step towards translating our findings into a therapeutic modality potentially useful for treating vascular disease.